General VirologyGeneral Virology

VIRUS STRUCTURE

Virion vs virusVirion vs virus

• Virion is the infectious particle

– composed of nucleic acid, protein capsid, +/- envelope

– may be extracellular or intracellular

• Virus is any stage of infection

How do we know that NA is genetic How do we know that NA is genetic material?material?

Hershey-Chase Fraenkel-Conrat Experiment

TRANSFECTION EXPTS

TRANSFECTION FAILS FOR SOME VIRUSES

WHY?

CapsidCapsid

• Functions– Protection of NA– Attachment for naked

viruses– Enzyme

• Helical vs Icosahedral Symmetry - Why do most viruses look alike?

• Tobacco mosaic virus is a ssRNA virus composed of 6000 nucleotides. The capsid is made of 2100 copies of a single protein subunit that contain 158 amino acids. Calculate the percentage of the genome that is used for structure.

• Helical- one axis of symmetry down center

• Multiple structural units

How do helical viruses differ?How do helical viruses differ?

Icosahedral symmetryIcosahedral symmetry

• 20 identical equilateral triangles

• Structural units on faces to give morphological capsomers– Pentons (5 fold axis of

symmetry)– Hexons

• 3 fold through face• 2 fold through edge

How do spherical viruses differ?

EnvelopeEnvelope• Attachment

• Entry

• Assembly- matrix proteins

• Release

• Proteins are viral

• Lipids are host

• Rare in plants or bacteria - why?

• If the membrane envelope is destroyed, the virus becomes noninfectious. Why?

Herpesvirus complexityHerpesvirus complexity

• Tegument proteins - 12/84 viral proteins in HSV

• Potential role?

• Virion mRNA

– DNAase virion nucleic acids

– RT-PCR

– probe genome array

• Potential role?

Genome - Genome - DNA or RNA

• strandedness - (single) (double)

• linear or circular, partial double stranded circle

• number (single, segmented, multicomponent)

How do we experimentally show that DNA or RNA is the virus genetic material?

RNA GenomesRNA Genomes

• sense (positive-sense, negative-sense, ambisense)

• presence or absence of 5'-terminal cap or 5'-covalently-linked protein

• presence or absence of 3'-terminal poly (A) tract

• Retroviruses - replication strategy

Some viruses have high degree of secondary Some viruses have high degree of secondary structurestructure

• Poliovirus - 5’ internal ribosome entry site (IRES)

Guest et al. 2004. J. Virol. 78: 11097.

SARS/coronaviruses have conserved 3’regionSARS/coronaviruses have conserved 3’region

• SARS s2m in red

• a - green = 530 loop of 16S RNA

• Similar binding properties:

– b - blue = S12

– magenta = IF1

• Possible role for s2m

– Hijacks protein synthesis from cell(binding cell factors)

– Needed to bind to similar viral protein for transcription

• Potential drug target in red tunnelRobertson et al. 2005. PLOsBiology:3.

DNA Viruses may be large genomesDNA Viruses may be large genomes

• PolyDNAvirus (PDV) - contain many DNA segments

• Mimivirus - larger than small bacteria

Host-induced modificationHost-induced modification

• Viral property that varies depending on the host

• Phage DNA hydroxymethyl cytosine (HMC) replaces C – Viral enzymes: C to HMC– Viral DNA polymerase: adds

HMC not C– What is advantage of HMC?

• Glucose is attached to HMC– Host enzyme needed to

prepare glucose– Protects against host nuclease

• What would happen if virus without glucose enters host with RE?

• What would happen if virus with glucose enters host w/o enzyme to create UDP- glucose?Host enzyme makes

ProteinsProteins

• structural proteins • non-structural virion

proteins– transcriptase, – protease– integrase

How to identify virion proteinsHow to identify virion proteins

• Purify KSHV virions

• Run on SDS PAGE

• Excise bands, digest - get sequence and compare to database

Chemical synthesis of poliovirus: What are Chemical synthesis of poliovirus: What are the implications?the implications?

• Small genome positive strand RNA - sequence known

• Synthesized small DNA segments (~ 69 nucleotides) with overlapping complementary segments

• Added a T7 phage promotor to DNA

• Used DNA to make genome RNA in HeLa cell lysate with T7 polymerase

• Results: How do you show success?

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International Congress on Taxonomy of VirusesInternational Congress on Taxonomy of Viruses http://www.ncbi.nlm.nih.gov/ICTV/

• Morphology

– virion size

– enveloped or naked nucleocapsid

– capsid symmetry and structure

• Genome characteristics

• Replication strategy

• Antigenic Properties

Baltimore classificationBaltimore classification

WHY TRANSFECTION FAILS

ONE STEP GROWTH CURVEONE STEP GROWTH CURVE

• 1939- Ellis and Delbruck: • Infection with a high multiplicity

of infection (MOI): ratio of virus to host cell– Simultaneous infection – Single replication cycle

• Sample at time intervals by plaque count for plaque-forming units (PFU),

• Identification of latent phase• Determination of burst size/viral

yield

Measuring Intracellular EventsMeasuring Intracellular Events

• Sample at time intervals after lysing cells (1952 - Doermann)

– Chloroform

– Lysis from without

• Identification of eclipse and maturation phases

Maturation phase